Identification and Enzymatic Activity Evaluation of a Novel CYP2C9 Allelic Variant Discovered in a Patient

Warfarin is a widely prescribed anticoagulant but the doses required to attain the optimum therapeutic effect exhibit dramatic inter-individual variability. Pharmacogenomics-guided warfarin dosing has been recommended to improve safety and effectiveness. We analyzed the cytochrome P450 2C9 (CYP2C9) and vitamin K epoxide reductase complex subunit 1 (VKORC1) genes among 120 patients taking warfarin. A new coding variant was identified by sequencing CYP2C9. The novel A > G mutation at nucleotide position 14,277 led to an amino acid substitution of isoleucine with valine at position 213 (I213V). The functional consequence of the variant was subsequently evaluated in vitro. cDNA of the novel variant was constructed by site-directed mutagenesis and the recombinant protein was expressed in vitro using a baculovirus–insect cell expression system. The recombinant protein expression was quantified at apoprotein and holoprotein levels. Its enzymatic activities toward tolbutamide, warfarin and losartan were then assessed. It exhibited changed apparent Km values and increases of 148%, 84% and 67% in the intrinsic clearance of tolbutamide, warfarin and losartan, respectively, compared to wild-type CYP2C9*1, indicating dramatically enhanced in vitro enzymatic activity. Our study suggests that the amino acid at position 213 in wild-type CYP2C9*1 may be important for the enzymatic activity of CYP2C9 toward tolbutamide, warfarin and losartan. In summary, a patient taking high-dose warfarin (6.0 mg/day) in order to achieve the target international normalized ratio was found to have a mutation in the CYP2C9 gene.

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Mutational Analysis of Ocriplasmin to Reduce Proteolytic and Autolytic Activity in Pichia pastoris

Biological Procedures Online ◽

10.1186/s12575-020-00138-0 ◽

2020 ◽

Vol 22 (1) ◽

Author(s):

Roghayyeh Baghban ◽

Safar Farajnia ◽

Younes Ghasemi ◽

Reyhaneh Hoseinpoor ◽

Azam Safary ◽

...

Keyword(s):

Pichia Pastoris ◽

Proteolytic Activity ◽

Mutational Analysis ◽

Expression System ◽

Catalytic Efficiency ◽

Site Directed Mutagenesis ◽

Wild Type ◽

Vitreomacular Adhesion ◽

Autolytic Activity

Abstract Background Ocriplasmin (Jetrea) is using for the treatment of symptomatic vitreomacular adhesion. This enzyme undergoes rapid inactivation and limited activity duration as a result of its autolytic nature after injection within the eye. Moreover, the proteolytic activity can cause photoreceptor damage, which may result in visual impairment in more serious cases. Results The present research aimed to reduce the disadvantages of ocriplasmin using site-directed mutagenesis. To reduce the autolytic activity of ocriplasmin in the first variant, lysine 156 changed to glutamic acid and, in the second variant for the proteolytic activity reduction, alanine 59 mutated to threonine. The third variant contained both mutations. Expression of wild type and three mutant variants of ocriplasmin constructs were done in the Pichia pastoris expression system. The mutant variants were analyzed in silico and in vitro and compared to the wild type. The kinetic parameters of ocriplasmin variants showed both variants with K156E substitution were more resistant to autolytic degradation than wild-type. These variants also exhibited reduced Kcat and Vmax values. An increase in their Km values, leading to a decreased catalytic efficiency (the Kcat/Km ratio) of autolytic and mixed variants. Moreover, in the variant with A59T mutation, Kcat and Vmax values have reduced compared to wild type. The mix variants showed the most increase in Km value (almost 2-fold) as well as reduced enzymatic affinity to the substrate. Thus, the results indicated that combined mutations at the ocriplasmin sequence were more effective compared with single mutations. Conclusions The results indicated such variants represent valuable tools for the investigation of therapeutic strategies aiming at the non-surgical resolution of vitreomacular adhesion.

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Sortase mutants with improved protein thermostability and enzymatic activity obtained by consensus design

Protein Engineering Design and Selection ◽

10.1093/protein/gzaa018 ◽

2019 ◽

Vol 32 (12) ◽

pp. 555-564

Author(s):

Magdalena Wójcik ◽

Susana Vázquez Torres ◽

Wim J Quax ◽

Ykelien L Boersma

Keyword(s):

Staphylococcus Aureus ◽

Amino Acid ◽

Enzymatic Activity ◽

Sortase A ◽

Wild Type ◽

Protein Thermostability ◽

Consensus Analysis ◽

Gram Positive Bacteria ◽

Covalently Linked

Abstract Staphylococcus aureus sortase A (SaSrtA) is an enzyme that anchors proteins to the cell surface of Gram-positive bacteria. During the transpeptidation reaction performed by SaSrtA, proteins containing an N-terminal glycine can be covalently linked to another protein with a C-terminal LPXTG motif (X being any amino acid). Since the sortase reaction can be performed in vitro as well, it has found many applications in biotechnology. Although sortase-mediated ligation has many advantages, SaSrtA is limited by its low enzymatic activity and dependence on Ca2+. In our study, we evaluated the thermodynamic stability of the SaSrtA wild type and found the enzyme to be stable. We applied consensus analysis to further improve the enzyme’s stability while at the same time enhancing the enzyme’s activity. As a result, we found thermodynamically improved, more active and Ca2+-independent mutants. We envision that these new variants can be applied in conjugation reactions in low Ca2+ environments.

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Predicting Evolution by In Vitro Evolution Requires Determining Evolutionary Pathways

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.46.9.3035-3038.2002 ◽

2002 ◽

Vol 46 (9) ◽

pp. 3035-3038 ◽

Cited By ~ 37

Author(s):

Barry G. Hall

Keyword(s):

Amino Acid ◽

Site Directed Mutagenesis ◽

Dna Shuffling ◽

Directed Mutagenesis ◽

Amino Acid Substitutions ◽

Single Mutation ◽

Wild Type ◽

In Vitro Evolution ◽

Evolutionary Pathway

ABSTRACT In an early example of DNA shuffling, Stemmer (W. P. C. Stemmer, Nature 370:389-390, 1994) demonstrated a dramatic improvement in the activity of the TEM-1 β-lactamase toward cefotaxime as the consequence of six amino acid substitutions. It has been pointed out (B. G. Hall, FEMS Microbiol. Lett. 178:1-6, 1999; M. C. Orencia, J. S. Yoon, J. E. Ness, W. P. Stemmer, and R. C. Stevens, Nat. Struct. Biol. 8:238-242, 2001) that the power of DNA shuffling might be applied to the problem of predicting evolution in nature from in vitro evolution in the laboratory. As a predictor of natural evolutionary processes, that power may be misleading because in nature mutations almost always arise one at a time, and each advantageous mutation must be fixed into the population by an evolutionary pathway that leads from the wild type to the fully evolved sequence. Site-directed mutagenesis was used to introduce each of Stemmer's six substitutions into TEM-1, the best single mutant was chosen, and each of the remaining five substitutions was introduced. Repeated rounds of site-directed mutagenesis and selection of the best mutant were used in an attempt to construct a pathway between the wild-type TEM-1 and Stemmer's mutant with six mutations. In the present study it is shown (i) that no such pathway exists between the wild-type TEM-1 and the supereffective cefotaxime-hydrolyzing mutant that was generated by six amino acid substitutions via DNA shuffling (Stemmer, Nature 370:389-390, 1994) but that a pathway to a fourfold more efficient enzyme resulting from four of the same substitutions does exist, and (ii) that the more efficient enzyme is likely to arise in nature as the result of a single mutation in the naturally occurring TEM-52 allele.

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Mutational Analysis of Ocriplasmin to Reduce Proteolytic and Autolytic Activity in Pichia Pastoris

10.21203/rs.3.rs-62284/v1 ◽

2020 ◽

Author(s):

Roghayyeh Baghban ◽

Safar Farajnia ◽

Younes Ghasemi ◽

Reyhaneh Hoseinpoor ◽

Azam Safary ◽

...

Keyword(s):

Pichia Pastoris ◽

Mutational Analysis ◽

Expression System ◽

Catalytic Efficiency ◽

Site Directed Mutagenesis ◽

Wild Type ◽

Vitreomacular Adhesion ◽

Km Value ◽

Autolytic Activity

Abstract Background: Ocriplasmin (Jetrea) is using for the treatment of symptomatic vitreomacular adhesion. This enzyme undergoes rapid inactivation and limited activities duration as a result of its autolytic and proteolytic nature after injection within the eye. Moreover, the proteolytic activities can cause photoreceptor damage, which may result in visual impairment in the more serious cases.Results: The present research aimed to reduce the disadvantages of ocriplasmin using site-directed mutagenesis. To reduce the autolytic activity of ocriplasmin in the first variant, lysine 156 changed to glutamic acid and in the second variant for the proteolytic activity reduction, alanine 59 mutated to threonine. The third variant contained both the mutations. Expression of wild type and three mutant variants of ocriplasmin constructs were done in Pichia pastoris expression system. The mutant variants analyzed in silico and in vitro and compared to the wild type. The kinetic parameters of ocriplasmin variants showed both variants with K156E substitution were more resistant to autolytic degradation than wild-type. These variants also exhibited reduced Kcat and Vmax values. An increase in their Km values, leading to a decreased catalytic efficiency (the Kcat/Km ratio) of autolytic and mix variants. Moreover, in variant with A59T mutation, Kcat and Vmax values have reduced compared to wild type. The mix variants showed the most increase in Km value (almost 2-fold) as well as reduced enzymatic affinity to the substrate. Thus, the results indicated combine mutations at ocriplasmin sequence were more effective compared with single mutations. Conclusions: The results indicated such variants represent valuable tools for the investigation of therapeutic strategies aiming at non-surgical resolution of vitreomacular adhesion.

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Heterologous expression and characterization of wild-type and mutant forms of a 26 kDa endochitinase from barley (Hordeum vulgare L.)

Biochemical Journal ◽

10.1042/bj3220815 ◽

1997 ◽

Vol 322 (3) ◽

pp. 815-822 ◽

Cited By ~ 53

Author(s):

Mette D. ANDERSEN ◽

Arne JENSEN ◽

Jon D. ROBERTUS ◽

Robert LEAH ◽

Karen SKRIVER

Keyword(s):

Hordeum Vulgare ◽

Recombinant Protein ◽

Heterologous Expression ◽

Specific Activity ◽

Expression System ◽

Site Directed Mutagenesis ◽

Wild Type ◽

Hordeum Vulgare L ◽

Sequence Alignments ◽

Natural Form

To investigate structure–function relationships in plant chitinases, we have developed a heterologous expression system for the 26 kDa endochitinase from Hordeum vulgare L. (barley). Escherichia coli cells harbouring the gene in a T7 RNA polymerase-based expression vector synthesized completely insoluble recombinant protein under standard induction conditions at 37 °C. However, a concentration of soluble recombinant protein of approx. 15 mg/l was achieved by inducing bacteria at low temperature (15 °C). Recombinant endochitinase was purified to homogeneity and shown to be structurally and functionally identical to the seed protein. An average of three disulphide bonds are present in the recombinant enzyme, consistent with the number found in the natural form. The seed and recombinant proteins showed the same specific activity towards a high-molecular-mass substrate and exhibited similar anti-fungal activity towards Tricoderma reesei. Site-directed mutagenesis was used to replace residues that are likely to be involved in the catalytic event, based on structural similarities with lysozyme and on sequence alignments with related chitinases. The Glu67 → Gln mutation resulted in a protein with undetectable activity, while the Glu89 → Gln mutation yielded an enzyme with 0.25% of wild-type specific activity. This suggests that two acidic residues are essential for catalytic activity, similar to the situation with many other glycosyl hydrolases. Examination of conserved residues stretching into the proposed substrate binding cleft suggests that Asn124 also plays an important functional role.

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Importance of the content and localization of tyrosine residues for thyroxine formation within the N-terminal part of human thyroglobulin

Acta Endocrinologica ◽

10.1530/eje.0.1320611 ◽

1995 ◽

Vol 132 (5) ◽

pp. 611-617 ◽

Cited By ~ 10

Author(s):

Marcel T den Hartog ◽

Carin C Sijmons ◽

Onno Bakker ◽

Carrie Ris-Stalpers ◽

Jan JM de Vijlder

Keyword(s):

Amino Acid ◽

Medical Center ◽

Academic Medical Center ◽

Negative Control ◽

Site Directed Mutagenesis ◽

Wild Type ◽

Terminal Part ◽

Tyrosine Residues ◽

In The Wild

Den Hartog MT, Sijmons CC, Bakker 0, Ris-Stalpers C, de Vijlder JJM. Importance of the content and localization of tyrosine residues for thyroxine formation within the N-terminal part of human thyroglobulin. Eur J Endocrinol 1995;132:611–17. ISSN 0804–4643 Thyroxine (T4) is formed by coupling of iodinated tyrosine residues within thyroglobulin (TG). In mature TG, some iodinated tyrosine residues are involved preferentially in T4 formation. In order to investigate the specific role of various tyrosine residues in T4 formation, N-terminal TG fragments with mutated tyrosine residues were constructed. An N-terminal TG fragment 198 amino acids in size and containing seven tyrosine residues at amino acid positions 5, 29, 89, 97, 107, 130 and 192 was expressed in a baculovirus system. Using site-directed mutagenesis, eight mutant TG fragments were constructed in which different tyrosine residues were replaced by phenylalanine. In the first four TG mutants, one single tyrosine residue (5, 89, 97 or 130) was mutated. In the mutant Y(5,89,97,130)F all of these four tyrosine residues were replaced. The sixth mutant Y(29,89,107,130,192)F contained only tyrosine residues 5 and 97 and the seventh (Y(29,89,97,192)F) contained only tyrosine residues 5, 107 and 130. A TG fragment (Y(5,29,89,97,107,130,192)F) in which all tyrosine residues were replaced by phenylalanine was used as a negative control. After in vitro iodination with lactoperoxidase, specific T4 formation was established in the non-mutated wild-type N-terminal TG fragment. In general the T4 formation in the mutant TG constructs decreased when the total number of tyrosine residues in the 198 amino acid fragment decreased, except fragment Y(29,89,97,192) containing three tyrosine residues, two of them being 5 and 130. Although the rate of T4 formation in this mutated N-terminal TG fragment was lower, the ultimate T4 generation was the same as in the wild-type fragment. This indicates that a preferential involvement of tyrosines 5 and 130 in thyroid hormonogenesis may exist. JJM de Vijlder, Academic Medical Center, University of Amsterdam, Children's Hospital "Emma Kinderziekenhuis/Het Kinder AMC", PO Box 22700, 1100 DE Amsterdam, The Netherlands

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The 6th Amino Acid Mutation of Rep Protein Had No Effect on PCV2b but Enhanced PCV2d Virus Replication in Vitro

10.21203/rs.3.rs-202148/v1 ◽

2021 ◽

Author(s):

Xiaoyan Wu ◽

Shuo Wang ◽

Changxun Xin ◽

Chen Li ◽

Jianli Shi ◽

...

Keyword(s):

Amino Acid ◽

Viral Replication ◽

Porcine Circovirus Type ◽

Site Directed Mutagenesis ◽

Porcine Circovirus ◽

Wild Type ◽

Protein Amino Acid ◽

Rep Protein ◽

Significant Difference

Abstract Porcine circovirus type 2 (PCV2) is the etiological agent that primary cause of post-weaning multisystemic wasting syndrome (PMWS). The major genotypes, PCV2a, PCV2b and PCV2d, are highly prevalent, but now replaced with 2b and 2d in swine population in worldwide. Rep protein is the key protein for viral replication. Compared a large number of Rep protein amino acid (aa) sequences, we found that there were three sites with regular changes between 2b and 2d. In order to analyze the effect of key sites on viral replication, we used site-directed mutagenesis to mutate the 6th aa of Rep (alternations with asparagine and serine) between PCV2b and PCV2d, Two wild-type and two mutant viruses infectious clones were rescued by non-contaminated porcine kidney-15 (PK-15) cells. Real-time quantitative PCR and a one-step growth curve were used to determine viral load to assess the replication of rescued viruses. The results showed that there was no significant difference between the PCV2b mutation and the wild-type PCV2b virus in vitro, while the mutation ofPCV2d enhanced viral replication.

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Mutational analysis of ocriplasmin to reduce proteolytic and autolytic activity in Pichia pastoris

10.21203/rs.3.rs-62284/v2 ◽

2020 ◽

Author(s):

Roghayyeh Baghban ◽

safar farajnia ◽

Younes Ghasemi ◽

Reyhaneh Hoseinpoor ◽

Azam Safary ◽

...

Keyword(s):

Pichia Pastoris ◽

Proteolytic Activity ◽

Mutational Analysis ◽

Expression System ◽

Catalytic Efficiency ◽

Site Directed Mutagenesis ◽

Wild Type ◽

Vitreomacular Adhesion ◽

Autolytic Activity

Abstract Background: Ocriplasmin (Jetrea) is using for the treatment of symptomatic vitreomacular adhesion. This enzyme undergoes rapid inactivation and limited activity duration as a result of its autolytic nature after injection within the eye. Moreover, the proteolytic activity can cause photoreceptor damage, which may result in visual impairment in more serious cases.Results: The present research aimed to reduce the disadvantages of ocriplasmin using site-directed mutagenesis. To reduce the autolytic activity of ocriplasmin in the first variant, lysine 156 changed to glutamic acid and, in the second variant for the proteolytic activity reduction, alanine 59 mutated to threonine. The third variant contained both mutations. Expression of wild type and three mutant variants of ocriplasmin constructs were done in the Pichia pastoris expression system. The mutant variants were analyzed in silico and in vitro and compared to the wild type. The kinetic parameters of ocriplasmin variants showed both variants with K156E substitution were more resistant to autolytic degradation than wild-type. These variants also exhibited reduced Kcat and Vmax values. An increase in their Km values, leading to a decreased catalytic efficiency (the Kcat/Km ratio) of autolytic and mixed variants. Moreover, in the variant with A59T mutation, Kcat and Vmax values have reduced compared to wild type. The mix variants showed the most increase in Km value (almost 2-fold) as well as reduced enzymatic affinity to the substrate. Thus, the results indicated that combined mutations at the ocriplasmin sequence were more effective compared with single mutations. Conclusions: The results indicated such variants represent valuable tools for the investigation of therapeutic strategies aiming at the non-surgical resolution of vitreomacular adhesion.

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Single amino acid changes in the mumps virus haemagglutinin–neuraminidase and polymerase proteins are associated with neuroattenuation

Journal of General Virology ◽

10.1099/vir.0.009449-0 ◽

2009 ◽

Vol 90 (7) ◽

pp. 1741-1747 ◽

Cited By ~ 11

Author(s):

Tahir H. Malik ◽

Candie Wolbert ◽

Laura Nerret ◽

Christian Sauder ◽

Steven Rubin

Keyword(s):

Amino Acid ◽

Clinical Isolate ◽

Amino Acid Change ◽

Mumps Virus ◽

Site Directed Mutagenesis ◽

Single Amino Acid ◽

Supporting Evidence ◽

L Protein ◽

Single Amino Acid Change

It has previously been shown that three amino acid changes, one each in the fusion (F; Ala/Thr-91→Thr), haemagglutinin–neuraminidase (HN; Ser-466→Asn) and polymerase (L; Ile-736→Val) proteins, are associated with attenuation of a neurovirulent clinical isolate of mumps virus (88-1961) following serial passage in vitro. Here, using full-length cDNA plasmid clones and site-directed mutagenesis, it was shown that the single amino acid change in the HN protein and to a lesser extent, the change in the L protein, resulted in neuroattenuation, as assessed in rats. The combination of both amino acid changes caused neuroattenuation of the virus to levels previously reported for the clinical isolate following attenuation in vitro. The amino acid change in the F protein, despite having a dramatic effect on protein function in vitro, was previously shown to not be involved in the observed neuroattenuation, highlighting the importance of conducting confirmatory in vivo studies. This report provides additional supporting evidence for the role of the HN protein as a virulence factor and, as far as is known, is the first report to associate an amino acid change in the L protein with mumps virus neuroattenuation.

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